The gene CYP76B1 of a cytochrome P450 has been isolated from Helianthus tuberosus tuber. The expression of this gene in yeast has shown that it encoded an enzyme very actively catalyzing the O-dealkylation of various exogenous molecules with a high efficiency. It could, for this reason, be used as oxygenase, as biocatalyst either for the degradation of environmental pollutants or for the biosynthesis of organic compounds such as medicaments, perfumes or pigments.
The expression of CYP76B1 is strongly induced in plants brought into contact with certain exogenous metals or organic compounds. This induction may be exploited for the detection of environmental pollutants.
CYP76B1 metabolizes with high efficiency a wide range of xenobiotics, including alkoxycoumarins, alkoxyresofurins and several herbicides of the class of phenylureas. CYP76B1 also catalyzes the mono- and didealkylation of phenylureas such as the two herbicides chlortoluron and isoproturon, with turnover rates comparable to those reported for physiological substrates and produces non-phytotoxic compounds. CYP76B1 can therefore be used to alter the resistance of plants sensitive to this family of herbicides and for soil and groundwater bioremediation.
1. Field of Invention
The present invention relates to purified polypeptides and DNA sequences of CYP76B1, a cytochrome P450 molecule, which has been isolated from Helianthus tuberosus tuber, and a method for preparing the same. It further relates to methods for detecting environmental chemical pollution comprising determining the presence of CYP76B1 polypeptide. It furthermore relates to methods for producing transgenic plants incorporating a gene or a gene fragment CYP76B1 in a bioremediation perspective and for an agricultural use.
2. Description of the Related Art
Plants respond to environmental stress with a wide range of adaptative changes, including the induction of defense mechanisms. Plants defense against the attack from pathogens and predators, or against changes in environmental conditions like drought, salinity, increased UV light or extreme temperatures have been largely documented. Much less is known about plant response to chemical agression. Increasing amounts of pesticides and other industrial chemicals are introduced in the environment without real knowledge of their impact on plant metabolism, development and organoleptic properties. Evidence has been obtained that plant can accumulate, transform and store exogenous chemicals as conjugated, compartmented or bound residues (2, 3). The enzymatic equipment allowing plants to cope with the toxicity of xenobiotics is very similar to the enzymes involved in the metabolism of drugs in the liver of mammals (4, 5, 6, 7). It includes reductive, oxidative and hydrolytic enzymes, various transferases for the formation of conjugated metabolites, and systems for the regeneration of antioxidants like glutathione. The cytochrome P450s constitute one of the principal classes of enzymes responsible for this metabolism (12, 7). A characteristic common to both animal and plant enzymes is their inducibility by drugs and other exogenous toxins.
As in animals, cytochromes P450 form, in plants, the main class of oxidases involved in the metabolism of exogenous molecules, including herbicides and pollutants (8, 9, 10, 11, 12). They have been shown to catalyze hydroxylation, S-oxidation, N- or O-dealkylation of environmental chemicals, thereby increasing their hydrophylicity and allowing their subsequent conjugation or immobilization. Increased P450 content and enzymatic activities following treatment with metals or organic molecules including drugs, herbicides, herbicide safeners, solvents and industrial pollutants have been observed in many plant species (1, 11, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22). The induction of certain cytochrome P450s and of their enzymatic activities is currently used as an early marker of chemical pollution in marine environments in particular (46, 47, 26). Studies, which are very preliminary, have suggested that an induction of P450s may be detected in a polluted environment (47). In the case of the cinnamate 4-hydroxylase (C4H), increase in catalytic activity induced by chemicals was paralleled by an increase in the steady-state level of CYP73A1 transcripts (22).
7-Ethoxycoumarin is a synthetic fluorescent molecule, very similar to natural coumarins, widely used to assay the catalytic activity of cytochromes P450 involved in the metabolism of foreign compounds (23, 24). Increase in 7-10 ethoxycoumarin O-deethylase (ECOD) activity, fast and easy to measure, has often been taken as an index of the induction of these P450s in animals following environmental chemical stress (25, 26). P450-dependent ECOD activity was detected in several plant species (27, 28, 29). In both dicots and monocots, the activity was found strongly induced in response to exogenous chemicals (1, 27, 29). A more detailed analysis, performed on Helianthus tuberosus tuber, showed that the induction of ECOD activity was often several fold higher than that of a physiological activity like C4H (22). ECOD thus appeared as a good potential marker of chemical contamination. Fluorescence assay of ECOD activity is impossible in extracts from green plants (interference with chlorophyll).
Because of the enormous losses caused by indesirable plant growth, the problem of weed control is a major one in the agricultural economy. Herbicides are broadly used for controlling the growth of weeds in crops. Among herbicides, compounds show broad spectrum control, making them useful where complete eradication of vegetation is needed, and others show selective control with tolerance to agronomic crops. New methods for producing transgenic plants resistant to herbicides have been developed (EP 730030). These transgenic plants are transformed and generally express or overexpress an enzyme capable of detoxifying the chosen herbicide, making them resistant to that herbicide.
Among the herbicides or pesticides which have been developed, many of them are substitued urea or thiourea compounds, represented by the formula: R1R2Nxe2x80x94COxe2x80x94NR3R4 or R1R2Nxe2x80x94CSxe2x80x94NR3R4, and are used for destruction and prevention of weeds (WO 90/06680; WO 95/22547; U.S. Pat. Nos. 2,655,444; 2,655,447; 2,857,430; 3,912,496; 5,393,733; 5,512,535 FR 69 03 235; FR 71 06 517).
Most of these substituted urea or thiourea compounds have cyclic groups and amine radicals, which can be substituted with alkoxy radicals and/or alkyl radicals. Among them, the phenylurea class which can be represented by the formula: (substituted-phenyl)xe2x80x94NHxe2x80x94COxe2x80x94NR1R2, in which the phenyl group and the secondary amine radical can be substituted with alkoxy and/or alkyl radicals. This phenylurea class can be illustrated by chlortoluron (N-(3-chloro-p-tolyl)-Nxe2x80x2,Nxe2x80x2-dimethylurea) and by isoproturon (N-(4-isopropylphenyl)-Nxe2x80x2,Nxe2x80x2-dimethylurea).
The first coding sequence of plant P450 (CYP73A1) capable of metabolizing an herbicide (i.e. capable of hydroxylating chlortoluron) has been isolated in the laboratory (33). The reaction turnover is however too low (0.014 minxe2x88x921) for the enzyme to be capable of substantially increasing the resistance of plants to chlortoluron.
It has, moreover, been demonstrated that the resistance of plants to herbicides (chlortoluron* and chlorsulfuron) could be manipulated (increased or decreased) by genetic transformation using animal or bacterial P450 genes (48, 49, 50). No experiment of this type has been carried out up until now with plant genes. The work carried out with bacterial and animal genes strongly suggests, however, that it is possible to transfer the tolerance to herbicides from one plant to another.
The advantage of using plant genes is two-fold: i) the efficiency of the biotransformation with the plant P450 appears to be substantially greater than that obtained with the animal P450s tested, ii) the transformation of plants with plant genes is accepted better by public opinion.
The present invention is directed to purified CYP76B1 polypeptide and its biologically active fragments.
The invention further relates to nucleic acids fragments encoding the polypeptide or its biologically active fragments and specific oligonucleotides probes or primers which are obtained from these sequences.
A further object of the invention is to provide cloning and/or expression vectors comprising at least one of the said nuleic acid sequence, and host cells transformed with such cloning and/or expression vectors under conditions allowing the replication or/and the expression of said nucleic acid sequences. In another aspect, this invention provides methods for producing CYP76B1 recombinant polypeptides or their biologically active fragments by transformed host cells.
The invention is also directed to monoclonal and polyclonal antibodies or their fragments which are specific for the polypeptide defined above.
The invention further comprises methods for detecting abnormal expression or overexpression of the said polypeptide in biological plant samples comprising determining the accumulation of CYP76B1 polypeptides in accordance with immunological techniques or evaluating the accumulation of CYP76B1 mRNA transcripts accumulation in accordance with molecular hybridization techniques. The use of these methods for detecting environmental chemical pollution is also an aspect of this invention.
According to a further aspect, the invention relates to a method for producing organic compounds which are obtained by organic substrates biotransformation using the CYP76B1 oxygenase activities and a method for selecting organic compounds capable of being biotransformed by the polypeptides.
The present invention also concerns a method for screening organic compounds capable of being biotransformed by the CYP76B1 polypeptides oxygenase activities, especially for selecting herbicides or pesticides.
Furthermore the invention relates to a method for screening plant cells or plants which are resistant to a given herbicide or pesticide comprising transforming a plant cell or plant with a vector of the invention, testing the plant cell or plant for its ability to grow in the presence of normally inhibitory concentration of said herbicide or pesticide and selecting the plant cells or the plants which are resistant to the herbicide or to the pesticide.
The invention also comprises a transgenic plant having increased resistance to herbicides or pesticides wherein said plant is transformed by a vector according to the invention.
The invention finally relates to a method for the bioremediation comprising transgenic plants which express or overexpress the CYP76B1 gene.
The object of the present invention relates to a purified polypeptide which comprises an amino acid sequence selected from:
a) the amino acid sequence SEQ ID NO 1,
b) the amino acid sequence SEQ ID NO 2,
c) a fragment of the amino acid sequence SEQ ID NO 1 or SEQ ID NO 2 which retains at least one of their biological activities,
d) an amino acid sequence of a CYP76B1 polypeptide analogue.
In the description the following definitions are used:
CYP76B1 polypeptide or CYP76B1 protein: a polypeptide having amino acid sequence comprising all or part of the CYP76B1 or E3C amino acid sequences, respectively sequence SEQ ID NO 1 and sequence SEQ ID NO 2, or one of their fragments or derived fragments which are biologically active.
CYP76B1 analogue: a polypeptide or molecule which results from a genetic or chemical modification such as mutation, deletion, addition, substitution and/or chemical modification of at least one amino acid of the CYP76B1 polypeptide sequence, said polypeptide retaining at least one of the CYP76B1 polypeptide biological activities.
Biologically active: capable of providing enzymatic activity, especially oxygenase activities, and/or capable of being recognized by CYP76B1 polypeptide specific antibodies and/or capable of inducing antibodies which recognize these polypeptides.
Among the polypeptides of the invention, the polypeptide which has the amino acid SEQ ID NO 1 or SEQ ID NO 2, is preferred.
The invention further relates to a DNA sequence isolated encoding a polypeptide according to the invention.
In a preferred embodiment the invention relates to DNA sequence selected from:
a) DNA sequence SEQ ID NO 1,
b) DNA sequence SEQ ID NO 2,
c) a DNA sequence capable of specifically hybridizing with DNA sequence SEQ ID NO 1 or DNA sequence SEQ ID NO 2, or with their complementary sequence or their corresponding mRNA sequence,
d) a DNA sequence encoding a CYP76B1 analogue.
In a more preferred embodiment the invention comprises DNA sequence isolated of gene which encodes CYP76B1 or E3C polypeptide.
The different nucleic acid fragments of the invention can be obtained by screening cDNA or genomic DNA librairies using probes prepared from a DNA sequence encoding the CYP76B1 polypeptide. Such cDNA or genomic DNA libraries can be screened by standard molecular biological techniques. Oligonucleotide probes or primers which are capable of strongly and specifically hybridizing with any of the DNA sequences according to the invention, are included in the invention.
Oligonucleotides probes, primers or CYP76B1 polypeptides can also be obtained by chemical synthesis, and are included in the invention.
Preferably the probes or the primers according to the invention comprise at least 10 nucleotides and can be as long as the full-length sequence is encoding the CYP76B1 polypeptide. Among the shortest probes which contain about 10 to 20 nucleotides, the suitable conditions for hybridization correspond to stringent conditions which are normally used in standard methods, described for example in the experimental procedure. Preferentially the probes according to the invention are preliminary labeled before use, for example radio-labeled, chemiluminescent-labeled, fluorescent-labeled or enzyme-linked probes.
The present invention also comprises oligonucleotides primers which specifically hybridize with one of the DNA sequences according to the invention or which comprise the DNA sequence encoding one of the polypeptides and allows the amplification of the sequence by the PCR method or by one of its variants. Advantagously the oligonucleotide probes or primers comprise a sequence selected from:
a) sequence SEQ ID NO 3: 5xe2x80x2-ATATATGGATCCATGGATTTTCTTATAATAGTGAGTAC (sense),
b) sequence SEQ ID NO 4: 5xe2x80x2-TATATAGAATTCATGCTAGTTCAATGGTATTGGAACAACAC (reverse).
The DNA sequence according to the invention can be used for the preparation of CPY76B1 polypeptides or analogues. Such recombinant polypeptides can be produced by standard recombinant DNA techniques. Effective production system of recombinant polypeptides requires to have at one""s disposal a vector, i.e. plasmid or viral origin, and a compatible host cell. The vector comprises, at least a DNA sequence according to the invention under the control of element expression in the host cell. The host cell can be chosen among procaryote systems, such as bacteria, eucaryote systems, such as yeasts, in particular Saccharomyces cerevisiae, or plant cells, or any system which is advantageously available. Such vectors can be prepared in accordance with standard techniques, and the resulting clones can be introduced in the appropriate host cell by using well known methods such as electroporation.
The present invention comprises a vector for the cloning or/and the expression of a polypeptide according to the invention, containing a DNA sequence according to the invention.
In a preferred embodiment the vector is a plasmid vector.
The cells can be prepared by introducing a nucleotide sequence inserted in a vector, as defined above, into the host cells then these cells are cultured under conditions allowing the replication or/and the expression of transfected nucleic acid fragment. The invention also comprises host cell transformed by a vector according to the invention. Preferably the cell is Saccharomyces cerevisiae or a plant cell.
This invention further comprises methods for the production of a polypeptide comprising culturing cells according to the invention under conditions allowing the expression of polypeptide and recovering the said polypeptide from the culture medium. The polypeptide obtained by these methods, are also included in the invention.
The monoclonal or polyclonal antibodies or their fragments, labeled antibodies which specifically bind to a polypeptide according to the invention, are included in the invention.
Polyclonal antibodies can be prepared using standard methods from an animal which has been immunized against CYP76B1 polypeptide, produced for example by genetic recombination. Monoclonal antibodies can be prepared according to the standard method for culturing hybridomas described by Kxc3x6hler and Milstein (Nature 1975). Antibodies according to the invention or their fragments, such as Fab or F(abxe2x80x2)2 fragments, can be labeled or immuno-conjugated. The invention comprises a method for the purification or the detection of a polypeptide according to the invention comprising using an antibody according to the invention. Biochemical pollution marker comprising an antibody or an oligonucleotide probe according to the invention are also included in the invention.
The invention furthermore comprises method for detecting abnormal expression or overexpression of polypeptides according to the invention from a biological plant sample comprising bringing said sample into contact with an antibody according to the invention under conditions allowing the formation of an immunological complex between said polypeptides and said antibody, and detecting the formation of an immunological complex.
The invention also comprises a method for detecting abnormal expression or overexpression of polypeptides according to the invention from a biological plant sample comprising bringing said sample into contact with an oligonucleotide probe according to the invention under conditions allowing the formation of an hybridization complex between said oligonucleotide probe and an mRNA sequence encoding said polypeptide, and detecting the formation of an hybridization complex.
These methods more especially allow the detection of abnormal accumulation of CYP76B1 polypeptides or their mRNA transcripts in a biological sample, abnormal accumulation which is induced by metals or organic compounds. More generally, antibodies and probes according to this invention can advantageously be used in any situation where expression or overexpression of CYP76B1 polypeptides must be examined. The invention also comprises the use of these methods for detecting environmental chemical pollution.
The expression of the CYP76B1 polypeptide in yeast has shown that it encoded an enzyme very actively catalyzing the dealkylation of exogenous substrates such as alkoxycoumarin, alkoxyresorufin or phenylurea herbicides. The CYP76B1 polypeptide oxygenase activities can therefore be used for biosynthetizing organic compounds such as medicaments, perfumes or pigments. This invention comprises method for biosynthetizing organic compound comprising bringing an organic substrate of the polypeptide according to the invention into contact with said polypeptide under conditions allowing the synthesis of said organic compound by biotransformation of the substrate, and recovering said organic compound.
CYP76B1 polypeptide very actively catalyzes the O-dealkylation of various exogenous compounds and the mono- and didealkylation of herbicides of the phenylurea family, such as chlortoluron or isoproturon. CYP76B1 polypeptide very effectively attacks the N-dimethyl group which it oxidizes into an amine. All the phenylureas, and numerous other pesticides, carry this functional group and are therefore potential substrates of CYP76B1 polypeptide. Such a metabolism abolishes the phytotoxicity of these herbicides or pesticides.
This invention comprises a method for screening organic compounds capable of being biotransformed by a polypeptide according to the invention comprising bringing said organic compounds into contact with said polypeptide under conditions allowing the biotransformation of said organic compound, analyzing the obtained compounds and selecting the biotransformed organic compounds.
In a preferred embodiment, the organic compound is an herbicide or a pesticide.
CYP76B1 polypeptide can consequently be used to alter the resistance of plants sensitive to this family of herbicides or pesticides which are metabolized by the CYP76B1 oxygenase activities. Methods of transforming plant cells or plants with a nucleic fragment encoding a given polypeptide and a regulatory sequence providing expression of the given polypeptide in the plant cells are well known. The nucleic acid fragments according to the invention can be used to transform a wide variety of agricultural plants including dicotyledons such as tobacco, cotton, soybean, melon, and monocotyledons such corn, wheat or rice.
The invention comprises a method for screening plant cells or plant which are resistant to a given herbicide or pesticide comprising transforming a plant cell or plant by a vector according to the invention, testing the plant cell or plant for its ability to grow in the presence of a normally inhibitory concentration of said herbicide or pesticide and selecting the plants cells or the plant which are resistant to the herbicide or to the pesticide.
Preferably, said herbicide or pesticide is of the phenylurea family. In a more preferred embodiment said herbicide is the chlortoluron or the isoproturon.
The invention also comprises transgenic plant having increased resistance to herbicides or pesticides wherein said plant is transformed by a vector according to the invention.
The invention finally comprises a method comprising transgenic plant according to the invention for the bioremediation and transgenic plant according to the invention which is an agricultural plant.